Bioluminescent imaging of Borrelia burgdorferi in vivo demonstrates that the fibronectin‐binding protein BBK32 is required for optimal infectivity

Abstract

Summary The etiologic agent of Lyme disease, Borrelia burgdorferi, is transmitted via infected Ixodes spp. ticks. Infection, if untreated, results in dissemination to multiple tissues and significant morbidity. Recent developments in bioluminescence technology allow in vivo imaging and quantification of pathogenic organisms during infection. Herein, luciferase-expressing B. burgdorferi and strains lacking the decorin adhesins DbpA and DbpB, as well as the fibronectin adhesin BBK32, were quantified by bioluminescent imaging to further evaluate their pathogenic potential in infected mice. Quantification of bacterial load was verified by quantitative PCR (qPCR) and cultivation. B. burgdorferi lacking DbpA and DbpB were only seen at the 1 h time point post-infection, consistent with its low infectivity phenotype. The bbk32 mutant exhibited a significant decrease in its infectious load at day 7 relative to its parent. This effect was most pronounced at lower inocula and imaging correlated well with qPCR data. These data suggest that BBK32-mediated binding plays an important role in B. burgdorferi colonization. As such, in vivo imaging of bioluminescent Borrelia provides a sensitive means to detect, quantify, and temporally characterize borrelial dissemination in a non-invasive, physiologically relevant environment and, more importantly, demonstrated a quantifiable infectivity defect for the bbk32 mutant.

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region, Causing Fibronectin to Undergo Conformational Extension

“…The concentrations of proteins and the thermodynamic parameters are shown in Table 2. and mode of infection (20). Further, BBK32 is distinct from the streptococcal FN-binding proteins in that it lacks additional FNBRs and attaches to the bacterial cell surface by its N rather than its C terminus (22).…”

“…1). BBK32, an FN-binding protein (19) from the Lyme diseasecausing spirochete Borrelia burgdorferi, contributes to optimal infectivity as the infectious process progresses from colonization to dissemination (20). BBK32 has an FN-binding sequence that is non-repetitive (21) and also differs from FNBPA, SfbI, and FnZ in being linked to the bacterial cell surface by its N terminus rather than the C terminus (22).…”

BB0744 Affects Tissue Tropism and Spatial Distribution of Borrelia burgdorferi

“…These strains were additionally screened for the presence or absence of bb0744 by PCR product size and DNA sequencing as previously described (54). Once validated, strains SH100 and SH200 were transformed with pBBE22luc under kanamycin selection and transformants were screened by PCR for the presence of the shuttle vector (31). Strains were then evaluated for plasmid content, and only those that maintained the complete plasmid set of the parent strain were used.…”

“…The time in which lymph nodes, joints, and other connective tissues become infected is relative to their proximity to the inoculation site (31,37). B. burgdorferi may travel through the lymphatic system not only for dissemination but also to interfere with the im-mune response and gain a survival advantage for spirochetes infecting all tissues (39).…”

“…The results imply that the available route of dissemination of the spirochetes has an impact on the extent of infection. Previously, studies have focused primarily on the route of dissemination through either connective tissues or the bloodstream (13,(30)(31)(32)(33)(34)(35). BBK32 has been implicated as one of the proteins involved in blood vessel dissemination, and spirochetes have been visualized adhering to and transmigrating across blood vessel epithelial cell layers during infection (36).…”